Angelique Florentinus-Mefailoski

Enzyme linked immuno mass spectrometric assay (ELIMSA).

UNLABELLED A new technology termed ELIMSA combines the specificity and enzymatic amplification of Enzyme Linked Immunosorbent assay (ELISA) with the sensitivity and flexibility of mass spectrometry (MS). At present, substrates for the reporter enzymes horseradish peroxidase (HRP) or alkaline phosphatase (AP) yield colored, fluorescent or luminescent products. The central concept of ELIMSA is that the reporter enzymes HRP and AP yield products that ionize efficiently with a high signal to noise ratio that can be measured by mass spectrometry. The reporter enzymes HRP or AP may be covalently attached to a specific detection probe such as a protein or an antibody to bind their target analyte and then catalyze the rapid production of ionizable, small-molecules. The use of mass spectrometry to measure small molecule products may commonly reach femto to picomol amounts on the column with high signal to noise ratio. Mass spectrometry combined with the enzyme amplification in ELISA provides absolute sensitivity to detect attomol of PSA and was comparable to, or more sensitive, than radio immune assays and electrochemical detectors but with only existing reagents and equipment. ELIMSA permits monitoring of multiple substrates and products and provides comparison to absolute standards. BIOLOGICAL SIGNIFICANCE There is an urgent need to detect and quantify low abundance proteins such as hormones, chemokines, cytokines, and others that exist at attomolar concentrations under physiological conditions by ELIMSA. A sensitive method for the quantification of immunological assays is obtained by applying mass spectrometry to detect the products of the alkaline phosphatase (AP) and horseradish peroxidase (HRP) enzyme reactions. There are many molecules from human subjects or micro-organisms that are of great importance to medicine, industry, nutrition or the environment that need to be repeatedly analyzed and are often near to, or beyond, the edge of existing analytical technology. The presence of molecules in biological samples, industrial products or the environment may be detected by probes that bind to the target analyte. Combining the reporter enzymes from ELISA with sensitive liquid chromatography (LC), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) will permit the sensitive detection and quantification of the molecular probes by Enzyme Linked Immuno Mass Spectrometric Assay (ELIMSA). The flexibility and sensitivity of mass spectrometry to measure large numbers of compounds simultaneously should permit the quantification of multiple ELIMSA reactions at separate mass-to-charge (m/z) ratios. Hence ELIMSA and it variants should permit the rapid and simple detection and quantification of many molecules over the complete range of biologically important concentrations without the use of radiolabels using only existing antibodies, reagents and instruments. Antibodies coupled to reporter enzymes that are widely used in biomedical and environmental applications can now be detected and quantified using ultra sensitive mass spectrometry to create a sensitive and flexible ELIMSA system. Absolute standards of analytes or enzyme product may serve as a reference.

Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen.

There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and (13)C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal (13)C isotope PA dilution curves, (ii) internal (13)C isotope one-point calibration, (iii) external PA standard curve, and (iv) external (13)C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap.

OxLDL receptor chromatography from live human U937 cells identifies SYK(L) that regulates phagocytosis of oxLDL.

The binding and activation of macrophages by microscopic aggregates of oxLDL in the intima of the arteries may be an important step towards atherosclerosis leading to heart attack and stroke. Microbeads coated with oxLDL were used to activate, capture and isolate the oxLDL receptor complex from the surface of live cells. Analysis of the resulting tryptic peptides by liquid chromatography and tandem mass spectrometry revealed the Spleen Tyrosine Kinase (SYK), and many of SYKs known interaction network including Fc receptors (FCGR2A, FCER1G and FCGR1A) Toll receptor 4 (TLR4), receptor kinases like EGFRs, as well as RNA binding and metabolism proteins. High-intensity precursor ions (∼9*E3 to 2*E5 counts) were correlated to peptides and specific phosphopeptides from long isoform of SYK (SYK-L) by the SEQUEST, OMSSA and X!TANDEM algorithms. Peptides or phosphopeptides from SYK were observed with the oxLDL-microbeads. Pharmacological inhibitors of SYK activity significantly reduced the engulfment of oxLDL microbeads in the presence of serum factors, but had little effect on IgG phagocytosis. Anti SYK siRNA regulated oxLD engulfment in the context of serum factors and or SYK-L siRNA significantly inhibited engulfment of oxLDL microbeads, but not IgG microbeads.

A method for the extraction of the endogenous tryptic peptides (peptidome) from human EDTA plasma

The proteins identified from endogenous peptides agreed between serum versus plasma, and tryptic versus non-tryptic peptides, when collected by C18 alone and analyzed by liquid chromatography electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) including amyloids, apolipoproteins, haptoglobin, complements, fibrinogens, hemopexin, antitrypsin and alpha 2 macroglobulin. Precipitation of polypeptides from plasma in 9 vol of 100% organic solvent followed by stepwise extraction of the insoluble pellet with an increasing fraction of water identified thousands of proteins. A Coomassie-blue protein binding assay, and tricine SDS-PAGE, showed that Acetonitrile-Water (AH) resulted in a greater relative enrichment of low molecular weight plasma polypeptides than Acetonitrile-Methanol Water (AMH). A total of 905,386 MS/MS spectra greater than ~10,000 (E4) counts were correlated by X!TANDEM to a federated human protein library of 153,124 different protein sequences that resulted in 58,223 fully tryptic peptides from 3463 Gene Symbols of which 1880 had ≥ 5 independent peptides (p ≤ 0.00001). The results were filtered and organized in an SQL database for analysis using the generic R statistical analysis system. Cellular proteins including secreted and exosome proteins, signaling factors, nucleic acid binding proteins, metabolic enzymes and uncharacterized factors were observed with a significant enrichment of expected protein-protein interactions by STRING analysis.

Re-evaluation of the rabbit myosin protein standard used to create the empirical statistical model for decoy library searching

A Rabbit myosin standard, like that used to create the empirical statistical model, was randomly and independently sampled by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) with a linear quadrupole ion trap. The rabbit myosin protein standard appeared pure by SDS-PAGE and CBBR staining but showed many other proteins by silver staining. The LC-MS intensity from myosin and IgG samples were above the 99% safe limit of detection and quantification computed from 36 blank LC-ESI-MS/MS runs. The myosin contained ≤406 Gene Symbols, open reading frames or loci where 79 protein types showed ≥3 peptides from X!TANDEM. Myosins, actin, troponin, other proteins showed 95%-100% homology between the rabbit versus the human decoy library. The myosin protein complex from STRING was true positive compared to random or noise spectra MS/MS with a low type I error (p-value) and low FDR (q-value) computed in R. SDS-PAGE, Western blot, comparison to random and noise MS/MS spectra, X!TANDEM p-values, FDR corrected q-values, and STRING all agreed that the error rate of LC-ESI-MS/MS with a quadrupole ion trap is far below that assumed a priori by the design of the empirical statistical model for decoy library searching.